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. 1999 Dec 27;147(7):1443–1456. doi: 10.1083/jcb.147.7.1443

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9805090.f3a.jpg — Effects of deletions in PDI on transport through the secretory pathway. a, Wild-type and mutant cells were pulse-labeled for 10 min at 30°C with ³⁵S-methionine/cysteine, followed by chase incubations for the indicated periods of time. At each time point, cells were lysed and CPY immunoprecipitated. Proteins were resolved on a 7.5% SDS gel and visualized by PhosphorImaging. b, SICs were prepared from wild-type cells and PDI deletion mutants. In vitro translated, radiolabeled ppαf was translocated into the ER of these SICs, and cells were incubated in the presence of ATP, an ATP-regenerating system, and 120 μg/25 μl reaction yeast cytosol for the indicated periods of time at 24°C. Budded vesicles containing gpαf were separated from SICs by differential centrifugation; radiolabeled, glycosylated gpαf was isolated from vesicle and SIC fractions by ConA precipitation, and quantified by scintillation counting.

graphic file with name JCB9805090.f3b.jpg — Effects of deletions in PDI on transport through the secretory pathway. a, Wild-type and mutant cells were pulse-labeled for 10 min at 30°C with ³⁵S-methionine/cysteine, followed by chase incubations for the indicated periods of time. At each time point, cells were lysed and CPY immunoprecipitated. Proteins were resolved on a 7.5% SDS gel and visualized by PhosphorImaging. b, SICs were prepared from wild-type cells and PDI deletion mutants. In vitro translated, radiolabeled ppαf was translocated into the ER of these SICs, and cells were incubated in the presence of ATP, an ATP-regenerating system, and 120 μg/25 μl reaction yeast cytosol for the indicated periods of time at 24°C. Budded vesicles containing gpαf were separated from SICs by differential centrifugation; radiolabeled, glycosylated gpαf was isolated from vesicle and SIC fractions by ConA precipitation, and quantified by scintillation counting.